Intestinal health and metabolic balance of zebrafish (Danio rerio) fed on diets containing enzymatically hydrolyzed distiller’s dried grains with solubles (DDGS)

Distiller’s dried grains with solubles (DDGS) were subjected to compound enzymatic deep hydrolysis (CEDEH) and evaluated as a plant-protein substitute for fish meal in zebrafish diets. The CEDEH protocol sequentially applied cellulase and α-amylase followed by proteolysis using neutral and flavor proteases at a 2:1 ratio. Enzyme-treated DDGS were incorporated into experimental diets to partially replace fish meal at 0%, 10%, and 20% inclusion levels. Six dietary treatments were established: DNP10 and DNP20 (neutral protease–hydrolyzed DDGS at 10% and 20%), DFP10 and DFP20 (flavor protease–hydrolyzed DDGS at 10% and 20%), and DCP10 and DCP20 (mixed protease–hydrolyzed DDGS at 10% and 20%). A 30-day feeding trial was conducted on 2-month-old zebrafish to assess physiological responses, intestinal histology, digestive enzyme activity, and gut microbiome–metabolome responses. Notably, CEDEH processing significantly enhanced duodenal villus height (P < 0.01), indicating improved nutrient absorption capacity. At 10% substitution (DCP10 group), hepatic antioxidant capacity significantly improved, with superoxide dismutase (SOD) and glutathione (GSH) activities increasing by 24.8% and 90.8%, respectively, compared to controls (P < 0.05). Gut microbiota restructuring demonstrated functional probiotics enrichment, including Cellulomonas (+ 57.3%) and anti-inflammatory Tabrizicola (+ 354.33%). Concurrently, key digestive enzymes (amylase and trypsin) activities in the duodenum markedly increased by 32.1% and 27.4%. Metagenomic analysis revealed upregulation of key metabolic pathways (ko00020 TCA cycle; ko00290 branched-chain amino acid synthesis). Importantly, 20% substitution (DCP20) preserved intestinal integrity (goblet cells increased by > 40%) without compromising growth performance. These results confirm that CEDEH-treated DDGS is a suitable fish meal substitute at inclusion levels ≤ 20%, promoting host metabolism and disease resistance.